RYK is a unique member of the receptor tyrosine kinase (RTK) family and is a putative pseudokinase. It is part of a small but biologically significant group of RTKs that has a functionally inactive protein tyrosine kinase (PTK) domain, due to substitution of conserved residues required for tyrosine kinase activity. It binds to the Wnt family of ligands via its Wnt inhibitory factor (WIF) domain, and regulates important biological processes including cell differentiation, migration, and axon pathfinding and target selection.
RYK is a cell surface receptor with a single transmembrane domain, a small extracellular region and a tyrosine kinase-like domain located within the cytoplasm. The RYK extracellular domain has a single identified domain, the Wnt inhibitory factor (WIF) domain. This domain was originally described in the soluble WIF-1 protein, which carries out its biological functions by sequestering members of the Wnt or Hedgehog ligand families. RYK has previously been shown to function as a Wnt receptor, and recently to function in mammalian planar cell polarity (PCP) signaling pathways.
Progress in defining the biological role of RYK has trailed many of the other RTK members due to the unusual nature of the ligands and to RYK having no detectable kinase activity. The generation of RYK-deficient mice has shed some light on its functions. RYK−/− mice demonstrated a key role for RYK in craniofacial development and palate closure. RYK is required for neural progenitor cell differentiation into neurons and axon extension, and is critical for correct axon guidance in the developing nervous system. Importantly, RYK was shown to mediate Writ-induced axon repulsion. In rat models of spinal cord injury, injection of an anti-RYK polyclonal antibody prevented corticospinal tract axon retraction from the lesion, caused sprouting of axons at and caudal to the lesion, and enhanced functional recovery after injury.
Although RYK has an established role in the transduction of Wnt-initiated signals, elucidation of the exact mechanisms by which RYK functions at a molecular and cellular level has remained elusive. It has been shown that RYK can signal via the small GTPase RhoA, though the downstream mediators have not been identified. However, the effects of this pathway are unknown. Inhibiting RYK function with conventional PTK inhibitors has not been possible due to lack of intrinsic kinase activity. While some interactive partners have been identified, they have not yet provided a specific mechanism to antagonize RYK function. Attempts to generate inhibitory antibodies to RYK have been hampered by poor immunogenicity of the receptor extracellular domain, lack of characterized ligands, and lack of structural information as to how the receptor interacts with its ligands and co-receptors.
Previously, monoclonal antibodies to RYK were generated using a soluble version of the entire human RYK extracellular domain expressed in a mammalian expression system (Halford M M, et al., 1999, J. Biol. Chem. 274: 7379-7390). While some monoclonal antibodies were made to this region, they were of the IgM isotype, indicating a failure of the immune system to switch to the high-affinity IgG isotype, which is typically seen with antigens that poorly stimulate the immune response.
It has been previously demonstrated that RYK is processed in two steps. A first cleavage event removes the extracellular domain, while a second cleavage is catalyzed by the γ-secretase complex and acts to liberate the RYK intracellular domain (RYK-ICD) (Lyu J, et al., 2008, Dev Cell 15: 773-780).
The poor immunogenicity and the constitutive proteolytic processing of the RYK extracellular domain, and potentially reduced availability of the WIF domain associated therewith, have posed a challenge for the generation of inhibitory anti-RYK antibodies. Accordingly, there exists a need to provide a high affinity monoclonal antibody that binds human RYK, in particular the WIF domain of the extracellular domain of RYK.